Objective: To investigate the signaling pathway involved in the insulin-elicited anti-apoptotic effect during myocardial ischemia and reperfusion (MI/R) in vivo.
Methods: Male Sprague-Dawley rats were anesthetized and subjected to 30 min of myocardial ischemia followed by 4h-reperfusion. Rats were randomly treated with intravenous infusion of saline (vehicle, 4 ml.kg(-1).h(-1)), insulin (60 U/L), or insulin + wortmannin 5 min before reperfusion and continuing throughout the 4h-reperfusion period. Cardiac myocyte apoptosis was determined both qualitatively and quantitatively by DNA laddering and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) methods. Myocardial nitric oxide (NO) was measured by using NO-specific chemiluminescence detector. Activations of Akt and p38 mitogen-activated protein kinase (MAPK) were determined by kinase activity assays using corresponding kinase activity assay kits (Cell Signaling).
Results: In the vehicle-treated rats, MI/R caused significant cardiac myocyte apoptotic death. Treatment with insulin produced a significant anti-apoptotic effect as evidenced by a marked reduction of apoptotic index [(8.0 +/- 2.9)% vs. (19.3 +/- 4.6)% of vehicle, P < 0.01] and decreased formation of myocardial DNA fragmentation. In addition, insulin treatment produced 2.7-fold increase (P < 0.01) of myocardial Akt activity and 28% increase of myocardial NO production (P < 0.05), while p38 MAPK activity changed insignificantly as compared with that of vehicle (P > 0.05). Both insulin-induced Akt activation and anti-apoptotic effect could be abrogated by wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor.
Conclusion: In vivo treatment with insulin at the initial of reperfusion significantly reduced postischemic apoptotic death via the PI3-kinase-Akt signaling pathway. Akt, but not p38 MAPK, activation plays a key role in the insulin-induced anti-apoptotic effect in MI/R.